Effect Of Heat Shock Protein 27 On Homocysteine-induced Endothelial Injury And The Underlying Mechanism

Background:Atherosclerosis is a chronic inflammatory disease caused by immune regulation disorder and unbalanced lipid metabolism. It underlies the pathophysiology of coronary atherosclerotic heart disease.The endothelium is a physiologic barrier between circulating blood and vascular smooth muscle. It maintains vascular homeostasis through secreting various factors to balance vasodilatation and vasoconstriction under different stimuli. When this balance is broken, endothelial dysfunction occurs, causing damage to the vascular wall. Endothelial dysfunction is considered as an initiating factor and early marker for atherosclerosis. Early prevention and control of endothelial dysfunction is the key prevention of coronary heart disease prevention.Hyperhomocystinemia is recognized as an independent risk factor for the development of atherosclerosis. Evidences showed that elevation of plasma homocysteine (Hcy) could lead to endothelial dysfunction; this is the key of resulting in early atherosclerosis.Heat shock protein 27 (HSP27), a member of the small heat shock protein family, is involved in a wide variety of cellular processes such as apoptosis, inflammation, cell migration, and maintenance of arterial wall homeostasis. Studies show that HSP27 has positive effect against myocardial apoptosis, ischemia injury and inflammation; it can also modulate vascular smooth muscle cells proliferation and migration. Recent studies have found that HSP27 plays an important role in the development of atherosclerosis; HSP27 in atherosclerotic plaques diminishes with progression of the stage of the pathology. It is believed that the anti atherosclerosis effect of HSP27 may involve stabilizes fibrin clot, anti apoptosis, maintains vascular barrier function, stabilizes and polymerization of actins, inhibits monocyte/macrophage mediated cell damage. HSP27 may be a potential biomarker of atherosclerosis as well as a potential therapeutic target.Part I Hcy-induced endothelial cell injury and the related mechanismObjective:Elevation of plasma Hcy can lead to endothelial dysfunction, an early marker for atherosclerosis. Some studies have demonstrated that Hcy could activate mitochondrial pathway, which lead to inducing endothelial cell apoptosis. Death-associated protein kinase (DAPK), a known mediator of programmed cell death, is shown to be up-regulated in atherosclerotic lesions. DAPK could be activated by a variety of stimuli, serving as a converging point for apoptotic signaling. It was found that DAPK contributed to shear stress induced endothelial apoptosis. This study aims to investigate the endothelial apoptosis induced Hey in HUVECs and the regulatory effect of DAPK on Hcy-induced endothelial cell apoptosis.Methods:1. The effect of Hcy on cell viability in HUVECs was evaluated by MTT assays. Nuclei fragmentation observed by Hoechst 33342 staining. Flow cytometry was used to detect the apoptotic rate of HUVECs by using Annexin V-FITC/PI staining.2. The mRNA and protein expressions of DAPK in Hcy treated HUVECs were measured by qRT-PCR and Western blot.3. Inhibited the DAPK expression using siRNA and tested by Western blot.4. The effect of DAPK on cell apoptosis rate was detected with by flow cytometry. The effect of DAPK on mitochondrial membrane potential (MMP) with Rh123 staining by flow cytometry.5. The expressions of mitochondrial apoptosis related proteins such as Bcl-2, Bax, caspase-3 and PARP were tested by Western blot.Results:1. Hcy decreased HUVECs survival and induces apoptosis in dose-dependent manner;2. Hcy increased DAPK expression at both the mRNA and protein levels in HUVECs in dose-dependent manner.3. Transfectated with DAPK siRNA could down-regulate the expression of DAPK in HUVECs。4. The knockdown of DAPK rescued the Hcy-induced cell apoptosis.5. The knockdown of DAPK could improve mitochondrial membrane potential, increase the expression of Bcl-2, reduce the expression of Bax, and reduce the caspase-3 and PARP cleavage.Part Ⅱ The protective effect of HSP27 against endothelial cell injury and the underlying mechanismObjective:Elevated plasma Hcy could lead to endothelial dysfunction and is viewed as an independent risk factor for atherosclerosis. HSP27, a small heat shock protein, is reported to exert protective against atherosclerosis. This study aims to investigate the protective effect of HSP27 against Hcy-induced endothelial cell injury and to determine the underlying mechanism.Methods:1. Measurement of HSP27 expression in Hcy-treated HUVECs by Western blot.2. Construction of the HSP27 over-expressing cell line and detection of HSP27 expression by using fluorescence microscopy, qRT-PCR, and Western blot.3. Measurement of nitric oxide (NO) concentration in the medium by using a Total Nitric Oxide Assay Kit. Measurement of mRNA expressions of endothelial molecules by qRT-PCR.4. The effect of HSP27 over-expressing on cell apoptosis rate was detected with AnnexinV-PI staining by flow cytometry.5. The effect of HSP27 over-expressing on the Hcy-regulated DAPK expression.6. The effects of HSP27 over-expressing on reactive oxygen species (ROS) and MMP were determinated by flow cytometry.7. The effects of HSP27 over-expressing on the expressions of mitochondrial apoptosis related proteins such as Bcl-2, Bax, caspase-3 and PARP were tested by Western blot.Results:1. Hcy increased HSP27 expression in HUVECs dose-dependent manner.2. The HSP27 over-expressing cell line could over-exprss HSP27 stably.3. HSP27 attenuated the effects of Hcy on the expression of NO and cytokines in endothelial cells.4. Protective effect of HSP27 on Hcy-induced apoptosis.5. Over-expression of HSP27 did not affect the Hcy-regulated DAPK expression.6. HSP27 attenuated Hcy-mediated ROS generation and MMP reduction.7. HSP27 could increase the ratio of Bcl-2/Bax and inhibit caspase-3 and PARP activity.Conclusions:1. Upregulation of DAPK contributes to homocysteine-induced endothelial apoptosis via the modulation of Bcl-2/Bax and actibaton of caspase-3.2. HSP27 plays a protective role against Hcy-induced endothelial injury through modulation of ROS production and the DAPK downstream mitochondrial caspase-dependent apoptotic pathway.